The overall goal of these continuing studies is to understand how endothelial nitric oxide synthase (eNOS) signaling is regulated in the pulmonary system with emphasis on its role in the perinatal period. We have recently demonstrated in vivo, that endothelin-1 (ET-1) acutely decreases nitric oxide (NO) generation in a mechanism that involves alterations in endothelial NO synthase (eNOS) activity, and that these interactions mediate dynamic changes in vascular tone following acute, surgically-induced, changes in PBF. Acute changes in pulmonary blood flow (PBF) in the perinatal period are an integral part of surgical repair of many congenital heart defects (CHD). In patients with single ventricle anatomy and physiology, the post-operative balance of blood flow to the lungs and body is often dictated by the resistances of the respective vascular beds. In fact, post-operatively the dynamic changes in vascular resistance can lead to significant morbidity and mortality, and much of our therapies are focused on altering pulmonary vascular resistance in attempt to optimize cardiac output while maintaining enough pulmonary blood flow to ensure adequate systemic oxygenation. Our new in vitro data indicate that ET-1-mediated activation of PKCd activity blocks the shear mediated increase in NO generation. Our preliminary data have also identified a previously undescribed mechanism by which ET-1 inhibits eNOS activity: decreasing ser1177 phosphorylation secondary to a catalase-mediated decrease in hydrogen peroxide (H2O2) that is regulated by PKCd activation. Based on these data, we hypothesize that alterations in NO-ET-1 interactions mediate the dynamic changes in pulmonary vascular resistance immediately following surgically induced changes in PBF in children with CHD. Further, we hypothesize that ET-1, via ETB receptor signaling, decreases NO signaling secondary to increased PKCd activity, thereby limiting the increases in PBF following surgery. Thus, in this competitive renewal, we will utilize an integrated physiologic, biochemical, cellular, and molecular approach, to investigate this hypothesis and its mechanisms. A better understanding of these mechanisms may improve peri- operative treatment strategies and reduce both short and long-term morbidity and mortality in children with CHD. PUBLIC HEALTH RELEVANCE: Acute changes in pulmonary blood flow (PBF) are an integral part of surgical repair of many congenital heart defects and in patients with single ventricle anatomy and physiology, the resistances of the respective vascular beds often dictate the post- operative balance of blood flow to the lungs and body. Normal pulmonary vascular tone is regulated by a complex interaction of vasoactive substances, such as NO and ET-1, that are produced locally by the vascular endothelium and we propose to investigate the role of NO, ET-1, and their interactions, in mediated the dynamic changes in vascular tone associated with acute changes in PBF. A better understanding of these mechanisms may improve peri-operative treatment strategies for children with congenital heart disease and have important clinical implications for thoracic surgery, transplantation, as well as systemic vascular biological disorders.